PROJECT ABSTRACT/SUMMARY Cells assemble functionally diverse actin cytoskeleton networks with distinct architectures and dynamics to drive fundamental processes such as polarization, endocytosis, motility and division. The specific characteristics of different actin filament networks (actin filament density, organization and dynamics) are determined through the coordination action of specific sets of actin binding proteins (ABPs) with complementary binding properties. Most investigations primarily focus on individual F-actin networks. However, this provides limited overall understanding of F-actin network organization and function because cells typically assemble and use multiple F-actin networks simultaneously within the same cytoplasm. Consequently, F-actin networks must self-organize from a common pool of shared actin monomers and overlapping sets of ABPs. We have predicted that there are important interactions (cross talk) between networks that are critical for their form and function. Our long-term goal is to discover the direct and indirect interactions between self-organized F-actin networks, which are critical for establishing their unique identities and functions within a common cytoplasm, and to determine the underlying molecular mechanistic principles that govern these interactions. We are investigating two major actin cytoskeleton self-organization questions. The first is to determine the mechanisms by which the size and density of F-actin networks are regulated by competition for a limiting amount of actin monomers (Aim I). Although unassembled G-actin was not thought to be limiting, we systematically showed that competition for G-actin helps control the size and density of competitive F-actin networks in fission yeast, and that the actin monomer protein profilin plays a major role in regulating competition for limiting G-actin. Our goal is to determine the underlying mechanism by which profilin and other ABPs contribute to the proper distribution of G-actin between functionally diverse actin cytoskeleton networks. The second is to determine how diverse F-actin networks acquire the specific set of ABPs whose complementary biochemical activities help define their form and function (Aim II). We will investigate the underlying intrinsic molecular mechanisms by which ABPs self-sort to particular F-actin networks within a common cytoplasm, including (1) the contribution of competition and cooperation between ABPs for associating with actin filaments, and (2) whether actin assembly factors initiate self-sorting by biasing the association of particular ABPs.